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Coronaviruses induce entry-independent, continuous macropinocytosis.

Freeman MC, Peek CT, Becker MM, Smith EC, Denison MR - MBio (2014)

Bottom Line: MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells.These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading.In this work, we show that CoVs induce a macropinocytosis late in infection that is continuous, independent from cell entry, and associated with increased virus titers and cell fusion.

View Article: PubMed Central - PubMed

Affiliation: mark.denison@vanderbilt.edu.

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MHV-induced macropinocytosis is associated with, but independent of, syncytium formation. (A) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell for 8 h, treated with PEG for 1 min, washed, and incubated for 3 h or transfected with VSV-G for 24 h. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Syncytia with ≤10 nuclei were analyzed. Data are represented as the mean ± the standard error of the mean of two replicates, each performed in duplicate. n = ≥30 cells per replicate. (B, C) Cells were mock infected or infected with MHV at an MOI of 1 PFU/cell. Anti-CEACAM blocking antibodies were added at 2 hpi, nanoparticles were added at 5 hpi, and cells were washed, fixed, stained, and imaged at 8 hpi. The number of nuclei per syncytium (B) and the percentage of cells with internalized nanoparticles (C) were measured. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. ***, P < 0.0001; **, P < 0.01; *, P < 0.05; n.s., not significant.
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fig6: MHV-induced macropinocytosis is associated with, but independent of, syncytium formation. (A) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell for 8 h, treated with PEG for 1 min, washed, and incubated for 3 h or transfected with VSV-G for 24 h. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Syncytia with ≤10 nuclei were analyzed. Data are represented as the mean ± the standard error of the mean of two replicates, each performed in duplicate. n = ≥30 cells per replicate. (B, C) Cells were mock infected or infected with MHV at an MOI of 1 PFU/cell. Anti-CEACAM blocking antibodies were added at 2 hpi, nanoparticles were added at 5 hpi, and cells were washed, fixed, stained, and imaged at 8 hpi. The number of nuclei per syncytium (B) and the percentage of cells with internalized nanoparticles (C) were measured. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. ***, P < 0.0001; **, P < 0.01; *, P < 0.05; n.s., not significant.

Mentions: The necessity for cleaved spike protein in macropinocytosis induction could be explained either by a requirement for spike protein-mediated cell-cell fusion or by a direct role for spike protein in macropinocytosis induction. To distinguish between these possibilities, we used two approaches: induction of syncytia by different methods and blockade of syncytium formation with antireceptor antibodies. To test the cell fusion hypothesis, cells were chemically treated with polyethylene glycol 1500 (PEG-1500) or transfected with the vesicular stomatitis virus G protein (VSV-G) (Fig. 6A) to induce cell-cell fusion and then incubated with nanoparticles. Both PEG-1500 treatment and VSV-G expression resulted in small syncytia with fewer than 10 nuclei. The nanoparticle uptake levels of syncytia with similar numbers of nuclei from MHV-infected cells, PEG-1500-treated cells, and VSV-G-transfected cells were compared. PEG-1500 treatment resulted in nanoparticle uptake greater than that of mock-infected cells but significantly less than that seen during MHV infection. Expression of VSV-G did not result in greater nanoparticle uptake than in mock-infected cells. We next blocked the interactions of MHV A59 spike protein with its cellular receptor, carcinoembryonic antigen (CEACAM), by adding anti-CEACAM blocking antibodies at 2 hpi and measured the effect on syncytium size and nanoparticle uptake (Fig. 6B). The anti-CEACAM antibodies resulted in a significant reduction in syncytial cell number and size but did not significantly decrease nanoparticle uptake by infected cells (Fig. 6C). Together, these results demonstrate that cleaved spike protein at the cell surface, and not cell fusion alone, is required to initiate and sustain macropinocytosis.


Coronaviruses induce entry-independent, continuous macropinocytosis.

Freeman MC, Peek CT, Becker MM, Smith EC, Denison MR - MBio (2014)

MHV-induced macropinocytosis is associated with, but independent of, syncytium formation. (A) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell for 8 h, treated with PEG for 1 min, washed, and incubated for 3 h or transfected with VSV-G for 24 h. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Syncytia with ≤10 nuclei were analyzed. Data are represented as the mean ± the standard error of the mean of two replicates, each performed in duplicate. n = ≥30 cells per replicate. (B, C) Cells were mock infected or infected with MHV at an MOI of 1 PFU/cell. Anti-CEACAM blocking antibodies were added at 2 hpi, nanoparticles were added at 5 hpi, and cells were washed, fixed, stained, and imaged at 8 hpi. The number of nuclei per syncytium (B) and the percentage of cells with internalized nanoparticles (C) were measured. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. ***, P < 0.0001; **, P < 0.01; *, P < 0.05; n.s., not significant.
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fig6: MHV-induced macropinocytosis is associated with, but independent of, syncytium formation. (A) DBT cells were mock infected or infected with MHV A59 at an MOI of 1 PFU/cell for 8 h, treated with PEG for 1 min, washed, and incubated for 3 h or transfected with VSV-G for 24 h. Nanoparticles were added 3 h prior to fixation, and cells were washed, fixed, stained, and imaged. Syncytia with ≤10 nuclei were analyzed. Data are represented as the mean ± the standard error of the mean of two replicates, each performed in duplicate. n = ≥30 cells per replicate. (B, C) Cells were mock infected or infected with MHV at an MOI of 1 PFU/cell. Anti-CEACAM blocking antibodies were added at 2 hpi, nanoparticles were added at 5 hpi, and cells were washed, fixed, stained, and imaged at 8 hpi. The number of nuclei per syncytium (B) and the percentage of cells with internalized nanoparticles (C) were measured. Significance was assessed by one-way ANOVA with Dunnett’s post hoc test. ***, P < 0.0001; **, P < 0.01; *, P < 0.05; n.s., not significant.
Mentions: The necessity for cleaved spike protein in macropinocytosis induction could be explained either by a requirement for spike protein-mediated cell-cell fusion or by a direct role for spike protein in macropinocytosis induction. To distinguish between these possibilities, we used two approaches: induction of syncytia by different methods and blockade of syncytium formation with antireceptor antibodies. To test the cell fusion hypothesis, cells were chemically treated with polyethylene glycol 1500 (PEG-1500) or transfected with the vesicular stomatitis virus G protein (VSV-G) (Fig. 6A) to induce cell-cell fusion and then incubated with nanoparticles. Both PEG-1500 treatment and VSV-G expression resulted in small syncytia with fewer than 10 nuclei. The nanoparticle uptake levels of syncytia with similar numbers of nuclei from MHV-infected cells, PEG-1500-treated cells, and VSV-G-transfected cells were compared. PEG-1500 treatment resulted in nanoparticle uptake greater than that of mock-infected cells but significantly less than that seen during MHV infection. Expression of VSV-G did not result in greater nanoparticle uptake than in mock-infected cells. We next blocked the interactions of MHV A59 spike protein with its cellular receptor, carcinoembryonic antigen (CEACAM), by adding anti-CEACAM blocking antibodies at 2 hpi and measured the effect on syncytium size and nanoparticle uptake (Fig. 6B). The anti-CEACAM antibodies resulted in a significant reduction in syncytial cell number and size but did not significantly decrease nanoparticle uptake by infected cells (Fig. 6C). Together, these results demonstrate that cleaved spike protein at the cell surface, and not cell fusion alone, is required to initiate and sustain macropinocytosis.

Bottom Line: MHV-induced macropinocytosis results in vesicle internalization, as well as extended filopodia capable of fusing with distant cells.These results indicate that macropinocytosis likely facilitates CoV infection through enhanced cell-to-cell spreading.In this work, we show that CoVs induce a macropinocytosis late in infection that is continuous, independent from cell entry, and associated with increased virus titers and cell fusion.

View Article: PubMed Central - PubMed

Affiliation: mark.denison@vanderbilt.edu.

Show MeSH
Related in: MedlinePlus